The present invention relates to a process for preparing ketocarotenoids by cultivation of genetically modified organisms which, compared with the wild type, have a modified ketolase activity, to the genetically modified organisms, and to the use thereof as human and animal foods and for producing ketocarotenoid extracts.
Carotenoids are synthesized de novo in bacteria, algae, fungi and plants. Ketocarotenoids, i.e. carotenoids containing at least one keto group, such as, for example, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin, are natural antioxidants and pigments produced as secondary metabolites by some algae and microorganisms.
Because of their coloring properties, the ketocarotenoids and especially astaxanthin are used as pigmenting aids in livestock nutrition, especially in trout, salmon and shrimp rearing.
Astaxanthin is currently prepared for the most part by chemical synthesis processes. Natural ketocarotenoids such as, for example, natural astaxanthin are currently obtained in small quantities in biotechnological processes by cultivation of algae, for example Haematococcus pluvialis or by fermentation of genetically optimized microorganisms and subsequent isolation.
An economic biotechnological process for preparing natural ketocarotenoids is therefore of great importance.
Nucleic acids encoding a ketolase and the corresponding protein sequences have been isolated from various organisms and annotated, such as, for example, nucleic acids encoding a ketolase from Agrobacterium aurantiacum (EP 735 137, Accession No. D58420), from Alcaligenes sp. PC-1 (EP 735137, Accession No. D58422), Haematococcus pluvialis Flotow em. Wille and Haematoccus pluvialis, NIES-144 (EP 725137, WO 98/18910 and Lotan et al, FEBS Letters 1995, 364, 125-128, Accession No. X86782 and D45881, Paracoccus marcusii (Accession No. Y15112), Synechocystis sp. Strain PC6803 (Accession No. NP—442491), Bradyrhizobium sp. (Accession No. AF218415) and Nostoc sp. PCC 7120 (Kaneko et al., DNA Res. 2001, 8(5), 205-213; Accession No. AP003592, BAB74888).
EP 735 137 describes the preparation of xanthophylls in microorganisms such as, for example, E. coli by introducing ketolase genes (crtW) from Agrobacterium aurantiacum or Alcaligenes sp. PC-1 into microorganisms.
EP 725 137, WO 98/18910, Kajiwara et al. (Plant Mol. Biol. 1995, 29, 343-352) and Hirschberg et al. (FEBS Letters 1995, 364, 125-128) disclose the preparation of astaxanthin by introducing ketolase genes from Haematococcus pluvialis (crtw, crtO or bkt) into E. coli. 
Hirschberg et al. (FEBS Letters 1997, 404, 129-134) describe the preparation of astaxanthin in Synechococcus by introducing ketolase genes (crtO) from Haematococcus pluvialis. Sandmann et al. (Photochemistry and Photobiology 2001, 73(5), 551-55) describe an analogous process which, however, leads to the preparation of canthaxanthin and provides only traces of astaxanthin.
WO 98/18910 and Hirschberg et al. (Nature Biotechnology 2000, 18(8), 888-892) describe the synthesis of ketocarotenoids in nectaries of tobacco flowers by introducing the ketolase gene from Haematococcus pluvialis (crtO) into tobacco.
WO 01/20011 describes a DNA construct for producing ketocarotenoids, especially astaxanthin, in seeds of oilseed crops such as rape, sunflower, soybean and mustard, using a seed-specific promoter and a ketolase from Haematococcus pluvialis. 
All the processes described in the prior art for preparing ketocarotenoids and, in particular, the processes described for preparing astaxanthin have the disadvantage that the transgenic organisms provide a large quantity of hydroxylated byproducts, such as zeaxanthin and adonixanthin, for example.